The most successful of all carbohydrate pharmaceuticals so far have been the carbohydrate-based antibacterial vaccines [48]. The basis of using carbohydrates as vaccine components is that the capsular polysaccharides and the O-specific polysaccharides on the surface of pathogenic bacteria are both protective antigens and essential virulence factors. The first saccharide-based vaccines contained capsular polysaccharides of Pneumococci: in the United States a 14-valent vaccine was licensed in 1978 followed by a 23-valent vaccine in 1983. Other capsular polysaccharides licensed for human use include a tetravalent meningococcal vaccine and the Vi polysaccharide of Salmonella typhi for typhoid fever. The inability of most polysaccharides to elicit protective levels of anti-carbohydrate antibodies in infants and adults with weakened immune systems can be overcome by their covalent attachment to proteins that confer T-cell dependent properties [49]. This principle has led to the construction of vaccines against Haemophilus influenzae b (Hib) [37] and in countries where these vaccines are routinely used, meningitis and other diseases caused by Hib have been virtually eliminated [50]. Extension of the conjugate technology to the O-specific polysaccharides of Gram-negative bacteria has provided a new generation of glycoconjugate vaccines that are undergoing various phases of clinical trials [51].
Cholera remains an important public health problem. The long-term control of cholera depends on good personal hygiene, uncontaminated water supply and appropriate sewage disposal. However, the improvement of hygiene is a distant goal for many countries. Thus the availability of an effective cholera vaccine is important for the prevention of cholera in these countries. Research on new cholera vaccines has mainly focused on oral formulations that stimulate the mucosal secretory immune system. Two oral cholera vaccines have been experimented with on large scale in humans.
The first vaccine, containing inactivated bacterial cells and the B-subunit of cholera toxin, was tested in Bangladesh from 1985 to 1989. This vaccine, according to the WHO, may prove useful in the stable phase of refugee/displaced person crises, especially when given preventively. The second vaccine is a live attenuated vaccine containing the genetically manipulated V. cholerae O1 strain CVD 103-HgR. Despite its efficacy in adult volunteers, results of a large-scale field trial carried-out in Indonesia for 4 years have shown a surprisingly low protection. Moreover, one of the safety concerns associated with live cholera vaccine is a possible horizontal gene transfer and recombination event leading to reversion to virulence. [52]
More recently, conjugates of V. cholerae O1 lipopolysaccharide with cholera toxin variants were prepared with an adipic acid dihydrazide linker. In Phase I studies, these conjugates elicited vibriocidal antibodies in human volunteers, with IgM levels comparable to, and IgG levels superior to, the Ig levels elicited by a cellular vaccine [10, 36, 38, 39]. Conjugation of the V. cholerae O139 CPS to tetanus toxoid, and inoculation of mice with the conjugate, has also been described by Morris et al. in U.S. Pat. No. 5,653,986.
Until 1992, V. cholerae serogroup O1 was recognized as the sole cause of cholera epidemics, whereas the non-O1 serogroups were associated with sporadic cases of gastroenteritis and extra-intestinal infections. In late 1992, the etiological agent of a massive cholera epidemic was identified as non-O1 V. cholerae serogroup O139. [53] This was the first reported instance of an encapsulated strain that caused epidemic cholera. [11]
The surface polysaccharide of V. cholerae O1 is a lipopolysaccharide (LPS), whereas V. cholerae O139, in contrast, has a capsular polysaccharide (CPS) composed of a hexasaccharide repeating unit, containing a trisaccharide backbone and two branches [3, 4, 8, 11, 16, 17, 19, 26, 28, 30, 31, 35, 38, 42, 43, 45]. The repeating unit contains two negatively charged groups, a galacturonic acid carboxyl group and a cyclic phosphate diester. This repeating unit is incorporated into the V. cholerae O139 lipopolysaccharide as well as the capsular polysaccharide, and it is possible that the V. cholerae O139 CPS is in fact a very high molecular weight LPS.
Passive immunization of mice with antiserum to the V. cholerae O139 capsular polysaccharide has been shown to protect against variants of V. cholerae O139, and it has been proposed that conjugates of the V. cholerae O139 capsular polysaccharide with cholera toxin or toxoid might be “worthy of further study” [38]. A potential live oral vaccine, comprising a non-pathogenic deletion mutant of V. cholerae engineered to express the V. cholerae O139 capsular polysaccharide and core-linked O-polysaccharide, has been described [62]. The vaccine elicited anti-CPS antibodies in rabbits, but neither animal protection studies nor clinical results have been reported to date.
It has been proposed that a critical level of serum IgG to the surface polysaccharides of V. cholerae O1 and V. cholerae O139 confers serotype-specific immunity to cholera [3, 7, 17, 24, 25, 28, 29-32, 38, 39, 43, 44]. It has also been proposed that the level of IgG, rather than the total level of vibriocidal antibodies may correlate more accurately with protection against cholera, because (1) synthesis of IgG is predictive of long-lived immunity, probably reflecting induction of T-helper cells to the antigen-specific B-cells, and (2) IgG antibodies penetrate into the extracellular spaces and interior of the small intestine more effectively than IgM. IgG directed to the O-specific polysaccharide of V. cholerae O1 or V. cholerae O139 could confer protective immunity to cholera by inactivating the inoculum on the intestinal mucosal surface.
Currently, vibriocidal antibody titers induced by vaccines are regarded as being predictive of therapeutic utility, at least for vaccines that have passed regulatory review: vibriocidal titer is the only serologic assay required by the U.S. Food and Drug Administration for licensure of new cholera vaccine lots. [61]
Previously described conjugates of the V. cholerae O139 CPS have not demonstrated the induction of adequate levels of IgG antibodies to provide reliable vaccines; accordingly there still remains a need for improved conjugates.